Aims: Increasing post-flowering nitrogen (N) uptake is likely to improve seed N accumulation, ultimately leading to greater seed yield and N use efficiency (NUE). Methods: A comprehensive study on contrasting N utilization efficiency (NUtE) winter oilseed rape genotypes was conducted for three years (2017–2020) to unravel the effect of post-flowering N uptake on seed N accumulation. Results: Compared to the low NUtE genotype, the high NUtE genotype displayed respectively 31%, 28% and 70% greater root biomass, length and volume, along with 40%, 44%, 46% and 82% higher root nitrate reductase (NR), glutamine synthetase (GS), glutamate synthetase (GOGAT), and glutamate dehydrogenase (GDH) activities after flowering. These were accompanied by a significant increase (P < 0.05) in catalases (134%), peroxidase (45%), glutathione (45%) and ascorbate peroxidase (41%), leading to 27% higher seed N accumulation, 23% higher seed yield and 60% higher NUE. Conclusions: Stronger post-flowering N uptake potential for high NUtE genotype was a main contributor to the enhanced seed N accumulation, and ultimately increased seed yield and NUE. [ABSTRACT FROM AUTHOR]